review article developments in diagnosis of visceral...

Review ArticleDevelopments in Diagnosis of Visceral Leishmaniasis inthe Elimination Era

Om Prakash Singh and Shyam Sundar

Infectious Disease Research Laboratory, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University,Varanasi 221005, India

Correspondence should be addressed to Shyam Sundar; [email protected]

Received 16 October 2015; Revised 6 December 2015; Accepted 14 December 2015

Academic Editor: Barbara Papadopoulou

Copyright © 2015 O. P. Singh and S. Sundar. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.

Visceral leishmaniasis (VL) is the most devastating parasitic infection worldwide causing high morbidity and mortality. Clinicalpresentation of VL ranges from asymptomatic or subclinical infection to severe and complicated symptomatic disease. A majorchallenge in the clinical management of VL is the weakness of health systems in disease endemic regions. People affected byVL mostly present to primary health care centers (PHCs), often late in their therapeutic itinerary. PHC physicians face a majorchallenge: they do not deal with a single disease issue but with patients presenting with complaints pointing to several diagnosticpossibilities. Risk exists when some patients having less clinical manifestations are misdiagnosed. Therefore, field based accurate,sensitive, and cost effective rapid diagnostic tools that can detect disease in its mildest form are essential for effective control andreaching the goal of VL elimination. In this review, we discuss the current status and challenges of various diagnostic tools for thediagnosis of VL and assess their application in resource poor settings.

1. Introduction

Visceral leishmaniasis (VL) or kala-azar is one of the mostneglected poverty related disease with an estimated world-wide incidence of 0.2–0.4 million new cases per year [1].About 90%of these cases occur in just six countries, includingIndia, Bangladesh, Sudan, South Sudan, Ethiopia, and Brazil[1]. In the Indian subcontinent and East Africa, VL is causedby L. donovani, which is transmitted by the sand fly P.argentipes, without any known animal reservoir [2], whereas,in Europe, North Africa, and Latin America, it is causedby L. chagasi (syn. L. infantum) which have both caninesand human as reservoirs [3]. More than 100,000 cases occurin India alone every year and the state of Bihar accountsfor majority of these cases [4]. However, these figures areofficial report, mainly based on passive case reporting, andare considered to be an underestimation of the real numberof VL cases [5].

VL is clearly a poverty related disease, affecting indeedthe poorest of the poor but also blocking the economic devel-opment of affected areas. In the year 2000, Thakur describedthe socioeconomic conditions of a cohort of 938 VL patients

from Bihar in India. 75% of them were classified as poor(daily income < US $1) and 82% were engaged in agricultureand/or animal husbandry [6]. VL has recently earned mostpublic attention as one of the neglected diseases globally. Inthe Indian subcontinent (ISC), three countries affected byVL,India, Nepal, and Bangladesh, aspire to eliminateVL from thesubcontinent with a target of bringing down the incidenceof VL to < 1 per 10000 population by 2015 through variouscontrol measures [7, 8]. One of the important componentsin this endeavor is decreasing transmission through earlydiagnosis followed by complete treatment. However, whenthe control programs succeed in lowering the incidence ofthe targeted condition, the positive predictive values of thediagnostic tests decrease withmore risk of false positives. It istherefore important to ensure that the platform and format ofdiagnostic technologies are appropriate for the prevalence ofinfection in the local context. The most important challengewith the control approaches is its long-term sustainability.Sooner or later, primary care settings will need to be rein-volved because passive case finding can only be provided at alarge scale and in the long-term by the first-line care givers.

Hindawi Publishing CorporationJournal of Parasitology ResearchVolume 2015, Article ID 239469, 10 pageshttp://dx.doi.org/10.1155/2015/239469

2 Journal of Parasitology Research

In endemic areas of VL, however, clinical decisions takenby physicians are serial and dichotomized, meaning thatfor a given syndrome (e.g., fever) the possible diagnosesare explored step by step and with a “yes/no” approach.Hence, when clear signs and symptoms are present, a specificdisease is considered and when these are absent the diseaseis discarded and alternative diagnoses are sought. VL ischaracterized by a number of complexities, and its clinicalfeatures are often confused with other febrile illnesses. Riskof misdiagnosis may exist with patients having less clinicalmanifestations causing delay in treatment and thus leading tothe death of patients. To address this real challenge in clinicalcare settings, rapid and accurate confirmatory diagnostictest is needed as antileishmanial drugs can cause significantadverse reactions. Accurate diagnostic tools will have amajor impact on the ability of countries to estimate accuratedisease burden. It would also allow them to track diseasetrends over time and to determine the effectiveness of futurecontrol interventions such as improved diagnosis-treatmentalgorithms and new vector management strategies. In thefollowing sections, we briefly discuss the currently availablediagnostic approaches for VL along with their effectivenessand limitations at the primary health care facilities in diseaseendemic areas.

2. Current Diagnosis of VL and Challenges

VL is characterized by a persistent febrile syndrome, usuallyassociated with splenomegaly that progressively leads towasting, anaemia, anddeath due to bleeding or superimposedbacterial infection. Early detection and proper managementare crucial for control of this disease. Noninvasive rapid testto be used to diagnose VL and/or as a marker of cure atperipheral health centers could have a great impact on theway VL is managed in endemic communities. A comparativeoverview of sensitivities and specificities of various diagnosticassays currently in use for VL is presented in Table 1.

2.1. Parasitological Diagnosis: Most Specific (Gold Standard)but Less Sensitive. Detection of a parasite is a very specificmethod and is the first-line approach in VL diagnosis.Microscopic examination of peripheral blood smear or buffycoat is noninvasive first-line test. In the case of negativeresult, the same procedure is being done on splenic biopsiesor bone marrow and remains the most specific method ofdiagnosis still in practice in the Indian subcontinent and EastAfrica [9]. Parasitological test with bone marrow aspirate ismost frequent in Brazil [10]. However, these procedures areuncomfortable, are potentially dangerous, require consider-able skill, and thus are not practical at PHC level. In vitroculture of tissue aspirates or blood cells have shown up to100% sensitivity [11, 12], but these methods are expensive,time consuming, very tedious, and restricted to only dedi-cated research laboratories. Importantly, parasitological testis the only confirmatory test which exists for relapse patients.

To increase the sensitivity of parasitological diagnosis,antibodies conjugated with fluorescent against surface anti-gens of the parasite have been carried out in endemic settingsin Brazil, Spain, Tunisia, Italy, and Iran [3]. Requirement of

a fluorescence microscope restricts the use of IFAT test toreferral hospitals.

2.2. Immunological Diagnosis: Noninvasive and Rapid.Immunological diagnoses are based on the detection ofeither leishmania antigens or antileishmanial antibodiesin the blood samples. Several serological tests have beendeveloped for VL to replace parasitological methods andhave been evaluated in different endemic regions withvariable results (Table 1). Sensitivity and specificity ofsuch tests depend on the antigens (Figure 1), and, amongthese, rk39 ELISA and direct agglutination test (DAT) havebeen extensively validated in endemic areas to documentL. donovani infection and recommended for VL controlprograms [13]. However, in Kenya, VL policy specifies thatall serologically proven leishmaniasis is confirmed by spleenaspirate, a procedure that can only be performed in referralhospitals [14].

2.2.1. Direct Agglutination Test (DAT). DAT is a semiqual-itative test and highly adopted in field settings that usesmicroplate with V-shaped well in which coomassie stainedwhole promastigotes antigen is mixed and serially dilutedwith patient’s serum or blood. If specific antibodies arepresent, agglutination can be seen after 18 hrs (overnightincubation) with naked eye. DAT has been validated inseveral countries including India, Nepal, Bangladesh, Sudan,Ethiopia, Kenya, and Brazil [44]. Sensitivity and specificityof DAT vary from 70.5 to 100% and 53 to 100%, respectively[3, 44]. In a recent longitudinal study from India andNepal, strong associations were found between progressionto clinical VL and seropositivity or seroconversion with highDAT titer [13]. Overall, performance of DAT as a diagnostictest is satisfactory, economic (US $1.5–2.5 per test), andindependent of the geographical regions [45]. Requirementsof electricity, storage of antigens at 2–8∘C, multiple pipetting,and need of skilled personnel make it impossible to conductsuch diagnostic tests at PHCs.

2.2.2. ELISA. Detection of antileishmanial antibodiesthrough ELISA is very common, and its sensitivity/specificitymainly depends on the antigen used. Previously, ELISA withcrude or soluble antigens of promastigotes or amastigoteswas used, but cross reactivity was common resultingin giving it least priority in diagnosis. With the adventin technology, several recombinant antigens have beenmade in VL diagnosis with rK39 being on the top of allrecombinant antigens (sensitivity: 67–100%; specificity:93–100%). However, due to its low sensitivity in Africa [22],new generation fusion antigen k28 was developed withimproved sensitivity (92–100% in Sudan) [22] without anychanges of its sensitivity in the Indian subcontinent [46].rK28 ELISA is also useful in diagnosis of cutaneous VL inBrazil [47]. Most importantly, common drawback for allantibody-based detection systems including ELISA is thatantileishmanial antibodies persist for 16 years after completetreatment and thus cannot be used as test of cure or relapse[48]. Another limitation of ELISA is that it can be only done

Journal of Parasitology Research 3

Table 1: Sensitivity and specificities of various tests in VL diagnosis.

Methods Sample used Test time Skill level required Sensitivity(%)

Specificity(%) References

Parasitologicaldiagnosis

Splenic aspiration Spleen tissue Hours High 93–99 100 [15]Bone marrowaspiration/biopsy Bone marrow Hours High 53–86 100 [16, 17]

Lymph nodeaspiration Lymph Hours High 53–65 100 [3, 16]

Culture Spleen or bone marrow Days Medium 97–100 100 [11].

Immunologicaldiagnosis

IFAT Serum/plasma Hours High 80–100 96–100 [18]Direct agglutinationtest Serum/plasma Days Medium 94.80 97.10 [13, 19–21]

ELISA Serum/plasma Hours Medium 93–100 97–98 [21, 22]Saliva Hours Medium 83.30 88.6–100 [23]

Immunochromaticstrip test

Serum Minutes Low 96.3–100 90.1–100 [24–26]Blood Minutes Low 96–100 90.8–100 [25, 26]Saliva Minutes Low 82.50 84.6–91.48 [23]Urine Minutes Low 96.40 66.2–100 [27–29]

Immunoblottingassay Serum/plasma Hours Medium 83–94% 90% [30]

IFN-𝛾 release assay(IGRA) Whole blood Days Medium 80–85 100 [31]

KAtex test (KAtex) Urine Hours Medium 48–87 89.00–100 [32, 33]

Moleculardiagnosis

PCR

Whole blood Hours High 70–100 85–99 [34]Buccal swab Hours High 83.00 90.56 [35]

Urine Hours High 88.0 100 [36]Bone marrow Hours High 95.30 92.60 [37]

PCR ELISA Whole blood Hours High 83.90 100 [38]qPCR Whole blood Hours High 91.3–100% 95.0–100% [39]

Oligo C-testWhole blood Hours High 96.2 90.0 [40, 41]Lymph node Hours High 96.8 NA [40]Bone marrow Hours High 96.9 NA [40]

LAMP Whole blood Hours Medium 83.0 98.0 [42]Buffy coat Hours Medium 90.7 100 [43]

in research labs or well equipped hospitals and thus cannotbe practical at field setting in endemic areas.

2.2.3. RapidDiagnostic Test (RDT). RDTbased on the recom-binant K39 protein antigen is available and is reproducible,is economic (∼US $1.0 per test), is easy to perform, and cangive the results within 10 minutes [9, 49–51]. Sensitivity andspecificity of rK39 RDT are high in most of the endemicregions (Figure 1). Use of the rK39RDT is now recommendedin combination with a clinical case definition, as a positiverK39 RDT in a healthy subject is not diagnostic of acutedisease [52]. In India, about 15–32% of healthy individualsliving in the endemic region test positive with the rapid rK39strip test [52], which is a major drawback of this test as non-VL patients with mimicking illnesses like malaria, entericfever, and so forth might receive antileishmanial treatment.In India, VL elimination initiative has adopted the rK39 RDTas the main tool, but its limitations are sorely felt [52, 53].

Response of rK39 RDT is less effective in East Africa (Sudanand Ethiopia) demonstrating sensitivities between 70% and94% [22]. The rK39 strip test performs moderately in SouthAmerican region (Brazil and Venezuela) where sensitivitiesand specificities vary from 86 to 100% and 82 to 100%,respectively. Recently, WHO conducted the evaluation offive different immune-chromatographic tests (ICT) utilizingeither rK39 or rKE16 on the Indian subcontinent, East-Africa,and Brazil. Result of this study showed the sensitivity between36.8 and 100% and specificity between 90.8 and 100% [24].No test was a clear winner across all regions and conditions,but high diagnostic accuracy was shown by rk39 RDT inIndia and Nepal. Later on, in two subsequent separate studiesin India comparing the performance on serum and blood,k39 RDT showed excellent agreement with high diagnosticaccuracy [25, 26]. Importantly, in areas where K39 dipsticksare not readily available and parasitological diagnosis (e.g.,spleen aspiration) is not feasible, clinicians still tend to rely


after complete treatment:

Treatment At the end of the treatment:

hospital for clinical confirmation of disease

No VL(confirmed by physician)

Healthy(all test negative)

(34.78%)

Status of diagnostic tests in relapse:

(85.2%)

marrow (96.6% and 96.6%)

Status of diagnostic tests in HIV-VL coinfection:

Serological test: DAT (84% and 90%);

Molecular tests: PCR (whole blood 92% and 96%; bone marrow 98%), buffy coat (93.1% and 96.9%), and bone

HIV coinfection

ELISA (66% and 90%); western blot (84% and 82%); IFAT (11–82% and 93.0%)

: LST (−ve)−ve)

= +ve

Status of diagnostic tests in active VL:

83–94%(92.8–100% and 96–100%), and western blot (

70.5–100% and 53–100%),

93–99% and 100%) and (53–86% and 100%)BM

rK39 ELISA (99.6% and 96.5–100%), rK28ELISA (99.6% and 94.17–100%), rk39 RDT

and 90%)

qPCR +ve

Status of diagnostic tests in patients

western blot (+ve)ELISA +ve, rK28 ELISA +ve, and

89%), rK39= −ve

−ve and qPCR −ve

If fevers are >14 days,then do rk39 dipstick (ICT for VL) and parascreentest (for malaria)

becomes +ve,patients are referred to

Follow-up

(complete cure)

Status of diagnostic tests in asymptomatic infection: Status of diagnostic tests in cured VL:

(5.4–36.67), rk28 ELISA (6.8%) rKE16 RDT4.4–23%), rK39 ELISA

= NA

rk39 RDT (17.4%),4.5–9.7%), and (7.9–29%), and qPCR +ve, and qPCR +ve

+ve, rK28 ELISA +ve, and IgG1 ELISA

23%)blot (11.1–33.3%)

(86.3–97%), rK26 ELISA (<50%), and93.5%), rK39 ELISA

= NA

70–100% and 85–99%) andMolecular

Note: if k39 test

Natural host

Proven relapse(by physician) No relapse

(cured VL)

Asymptomatic orsubclinical infection

negative If k39 test becomes

suspicion of relapse during FUBM/spleen aspiration in case of clinical

Proven VL case(spleen size, blood chemistry, etc., by physician at hospital)

Parasitological tests: splenic/BM

= +veParasitological tests: splenic/BMParasitological tests: splenic/BM

assay: PCR (


Immunological tests: LST (40.3%) and IGRA

Molecular tests: PCRImmunological test: LST (


Molecular tests: PCRMolecular tests: PCR ( Molecular tests: PCR (

Parasitological tests: splenic (

+ve, rK39 ELISA Serological test: DATSerological test: DAT ( Serological test: DAT (

Serological test: DAT (

Serological test: DAT (

Immunological test: LST (21%) and IGRA (24%)

Immunological test

western (+ve)

Figure 1: Generalized scheme of disease identification in endemic areas and sensitivity of various diagnostic tests in different pathologicalcondition of VL. +ve: positive; −ve: negative; BM: bonemarrow; DAT: direct agglutination test; ELISA: enzyme linked immunosorbent assay;HIV: human immunodeficiency virus; IGRA: interferon-𝛾 release assay; LST: leishmanin skin test; NA: not applicable; qPCR: quantitativereal time PCR; PCR: polymerase chain reaction; VL: visceral leishmaniasis. Note: in the case of VL and HIV-VL coinfection, both sensitivityand specificity of the diagnostic tests are presented.

on clinical diagnosis with consequent overdiagnosis andmisallocation of already stretched resources.These RDT testshowever cannot discriminate between current, subclinical, orpast infections and are useless for diagnosis of relapses and asprognostic (cure) tests.

More recently, rK39 RDT was tested and evaluated onurine samples in India and Bangladesh with sensitivity of96.1–100% and 95%, respectively [27–29, 54]. Excellent diag-nostic performance of rK39 RDT was also shown with salivasamples from India and Tunisia [23, 55]. Performance of rK39strip test in HIV positive and parasitological confirmed VLpatients was also tested, which showed 77% sensitivity [56].

More extensive studies are needed to establish the rK39 RDTfor diagnosis of VL using saliva or urine samples and make italso practical in the field condition for the diagnosis of HIV-VL coinfection.

2.2.4. Latex Agglutination Test (KAtex). An antigen detectiontest would in theory be considered more specific thanantibody-based serological tests [57]. Antigen detection diag-nostic tools are required as a means of identifying symp-tomatic infections in immunocompetent and immunocom-promised patients (e.g., diagnosis of primary VL in Sudan,where rK39 RDTs lack sensitivity; diagnosis of relapse cases)


and as an indicator of cure. Antigenaemia is a predictablefeature of VL, and it is also demonstrated by the efficacy of theKAtex test to detect antigens in urine. KAtex is the only com-mercially available diagnostic test that has been developed fordetection of 5–20 kDa glycoprotein in the urine ofVLpatients[9]. KAtex is an easy, rapid, and field applicable test withhigh specificity; however, sensitivity was unsatisfactory andvariable in the studies conducted in Indian subcontinent andEast Africa [32, 58, 59]. KAtex is 85.7% sensitive in HIV-VLcoinfected patients and showed potential as prognostic test[60], but low specificity in immune-competent individuals isa major limitation [61]. The fact that KAtex is noninvasivetechnique and urine can be collected more easily than bloodmakes it more acceptable in nonsymptomatic individuals andwould allow a longer follow-up. However, its low sensitivityand requirement to boil the urine for five minutes to avoidfalse positive reactions which affects the reproducibility ofthis test limit the use of KAtex in peripheral health facilities.

2.2.5. Leishmanin Skin Test and Whole Blood Assay. Mon-tenegro test or leishmanin skin test (LST) measures thedelayed type hypersensitive reaction. It is very useful test inthe case with cutaneous leishmaniasis (CL) where healinglesions are primarily present. In the case with VL, it ismostly used along with serological markers in endemic areas.Though it has very little diagnostic value in VL (typically neg-ative due to anergic state), it is very useful inVL epidemiology[62]. A GMP-grade L. donovani antigen is unavailable, andintradermal administration followed by 48 hrs of test readingis unpractical at PHCs. Whole blood IFN-𝛾 release assays(IGRA) have recently been introduced as an alternative tothe LST, but clinical application of IGRA in VL diagnosis ortreatment is not yet fully established [31].

2.3. Molecular Diagnosis: Highly Sensitive and Specific.Although considerable scientific progress has been madeover the past decade including the genome sequencing ofL. donovani, these have not had any impact so far on thequality of clinical care for VL in the field. Though diagnosticaccuracy of molecular tests is excellent in laboratory basedevaluations, their cost and their clinical benefit when appliedin resource-constrained settings are still in debate. So far,various molecular detection methods targeting specific DNAand/or RNA genes have been developed [15]. Polymerasechain reaction (PCR) and real time quantitative PCR (qPCR)are most rewarding gene amplification technique beingrapid and quite sensitive but are not feasible in the fieldsettings. Furthermore, very few assays have been validatedon broad range of clinical samples, and none of them hasbecome a reference tool in VL diagnosis (reviewed in [63]).Nonetheless, it is currently not very clear how such innovativedevices can bemeaningfully applied within the health systemcontext of VL endemic areas. However, many researchersclaim that when VL control program succeeds in loweringthe incidence of infection, these molecular tests will be thenan instrumental in maintaining the sustainability of controlprogram by detecting the infection at very low levels.

3. Diagnosis of Asymptomatic orSubclinical Infection: A New Challenge inEndemic Areas

Chemotherapy alone as VL control tool is limited by the factthat only sick people will be treated. There are asymptomaticcarriers ranging between 1 : 2.4 in Sudan [64], 4 : 1 in Kenya[65], 5.6 : 1 in Ethiopia [66], 18 : 1 in Brazil [67], 50 : 1 in Spain[68], 4 : 1 in Bangladesh [69], and 8.9 : 1 in high-endemicvillages of India and Nepal [70, 71], constituting probableparasite reservoirs for sand fly vector (reviewed in [63, 72]).A recent mathematical model suggests a major role of theseasymptomatic infections in driving transmission of humanVL in ISC [73]. Strong evidence that these infections are realcomes from studies in which parasites could be cultured fromblood of healthy donors [74] or detected by PCR [19, 75].Diagnostic tests for asymptomatic infections are thereforeneeded in order to identify possible hotspots of transmissionin endemic areas. Invasive methods to demonstrate the pres-ence of parasites are unethical in asymptomatic individuals;there is therefore no gold standard. A number of studieshave used DAT or rK39 based serological tests to documentinfection; other studies have used seroconversion for oneor more tests as marker of infection [20]. Currently, statusof asymptomatic infection can be defined in several ways:culture positive, PCR and serology positive, or marker ofcellular immune response like IGRA test (Figure 1). More-over, there is little agreement between the different tests whenapplied cross-sectionally on the same population group [76].Serologic testing (e.g., DAT, rk39 ELISA, and western blotassays) is generally assumed to detect recent infection, butthe length of time serology remains positive, and whetherthis differs between VL patients and subclinically infectedindividuals (as seems likely based on the magnitude of thetiters) is not known with certainty. In the absence of a goldstandard it is hard to know whether these seropositives whoremained healthy were truly infected with L. donovani orwhether the serology results were just false positive results orprior infection that cleared [63]. The robustness of these testmeasures could also be influenced by other factors includinghandling variability and storage practices.Though serologicaltests are frequently used to measure L. donovani infections,there is little published data regarding their reproducibilitywhen applied to asymptomatic persons.

Xenodiagnosis is the most direct way and only proof ofstudy to ascertain the infectivity of such asymptomatic infec-tions in disease transmission (reviewed in [63]). Molina et al.used xenodiagnosis as a method to diagnose VL infection inHIV infected patients. Even asymptomatic patients in earlystages of HIV infection were able to infect sand flies [77]. In alater study they hypothesize that such patients could trigger anew transmission route of VL in Spain, where the natural hostof the disease is the domestic dog [78]. If asymptomaticallyinfected persons can also be a source of transmission thereis obviously a need to reconsider certain aspects of theVL control strategy. Therefore, more extensive studies arerequired in this area in order to provide the epidemiologicaland clinical evidence that could more broadly inform VL


control programs and facilitate the development of noveldiagnostic aids.

4. Diagnosis of PKDL: An Unresolved Mystery

In a common complication of VL, particularly in Sudan,and to a lesser extent Ethiopia, patients may develop achronic form called “post-kala-azar dermal leishmaniasis,”or PKDL [79], which occurs within weeks to a few monthsfollowing treatment, in up to 50–60% of people who haverecovered from VL. In the ISC, 10% of VL patients go on todevelop PKDL after an interval of 6 months to 4 years [80].Clinical features consist of hypopigmented macules and/ordiffuse infiltration, papules, nodules, or plaques and can beconfused with other skin disorders. Except from cosmeticdisfiguration, these patients do not suffer from any physicalhandicap. Although mortality from PKDL is low, PKDLpatients represent a largely neglected reservoir of infectionthat perpetuates anthroponotic L. donovani disease in India.Though PKDL in Sudan and in India are both due to L.donovani, Sudanese PKDL frequently self-heals (84% in 1 year[79]) whereas Indian PKDL takes longer time to self-heal.

The tools for diagnosis of PKDL are inadequate. Inendemic areas, clinical sign and symptoms, along with aprevious kala-azar episode and positive antibody tests (e.g.,rK39 RDT), are being used to diagnose PKDL without anyparasitological confirmation (reviewed in [81]). However,this approach may not be accurate enough as ∼10% ofPKDL patients have no history of VL and positivity ofserological tests up to several years after treatment [82]. Skinslit smear microscopy is the only confirmatory test but isvery painful and impractical with macular lesions [83]. LSThave low sensitivity, and it is hard to culture parasite dueto contamination [82]. Nested PCR is highly sensitive; andrecent development of kDNA based quantitative PCR hasshown to be efficacious in diagnosis of PKDL [84]. However,these molecular tests are very costly and need sophisticatedlaboratory to perform. There is a huge gap in treatment ofPKDL, and treatments with Ambisome have side effects [80].Therefore, rapid noninvasive point of care diagnostic tests isurgently required as focal VL outbreaks have been linked toan index case of PKDL [85].

5. Diagnosis of HIV-VL Coinfection:Time for Concerted Action

Theemergence ofHIV and its associationwithVLposes chal-lenges as how best to diagnose and treat patients presentingwith HIV-VL coinfection.The actual number of documentedcases of HIV-VL coinfection in India is underestimated dueto problems in recognition, reporting, and diagnosis. Patientswith HIV-VL coinfection represent an important but largelyneglected reservoir of parasites, and focal reemergence of VLhave been linked to an index case of HIV (reviewed in [86]).Sensitivity and specificity of various diagnostic methods forHIV-VL have been reviewed by Deniau et al. [87] and Cota etal. [88]. Serological assays are considered not accurate, sincethe majority of these patients often do not exhibit detectable

levels of antibodies. Parasitemia is higher in HIV coinfection[89], thus direct detection of parasite or its component inblood by PCR or qPCR is increasingly used not only fordiagnosis but also for the follow-up of the patients during andafter treatment, but these tests are often not readily availablein poor health care settings (reviewed in [88]). At present,there is not any clear evidence to support recommendationson serological or molecular diagnosis of HIV-VL (Figure 1).Consequently, diagnosis often relies on invasive spleen orbone marrow aspiration or with a combination of RDTs usedin a diagnostic algorithm.

6. Conclusion

The most important step in VL control is to knock out thelast case by employing effective strategies. This can be onlypossible with availability of rapid and cost effective diagnostictest in disease endemic regions in order to enable the physi-cians to make accurate therapeutic decisions as arsenal ofantileishmanial drugs is limited and is frequently associatedwith adverse events [90]. Diagnostics needs to be consideredbroadly and concerns a range of applications like infection,disease, severity, or response to treatment. Visualization ofamastigote in suspected VL using microscopy is still the clas-sical confirmatory diagnostic test for VL but is not practicalin the endemic areas. A number of less invasive serologicaltests like DAT, rK39 ELISA, and molecular tests, for example,PCR, are becoming more attractive now, but, at present,these tests are confined to the laboratory. rK39 dipstick orICT, despite the variability initially observed among differentproducers and countries, seems to be the first choice fordecentralized diagnosis of VL with a good sensitivity andspecificity. However, due to some limitations, it must be usedin combination with a standardized clinical case definition.No tests are currently available that can detect asymptomaticL. donovani infection or predict progression of infection toclinical VL disease. Diagnosis of HIV-VL coinfection will beanother problem in coming years. Last but not least, there is aclear need to bridge the gap between current practices in thefield of clinical management of VL and available technologyand research efforts for VL diagnostics.

Conflict of Interests

The authors declare that there are no competing interests.

Authors’ Contribution

Om Prakash Singh and Shyam Sundar conceived and wrotethe final version of the paper. No writing assistance wasutilized in the production of this paper.

Acknowledgments

This work was supported by Extramural Programme of theNational Institute of Allergy and Infectious Disease (NIAID),National Institute of Health (TMRCGrant no. P50AI074321),and Foundation for National Institute of Health (FNIH)through Bill & Melinda Gates Foundation.


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